- Email: [email protected]
Gene therapy targeted by ionizing radiation
0360-3016/92 $5.00 + .oO Copyright 0 1992 Pergamon Press Ltd.
Inr. J Rodrarion Oncology Rio/. Phys, Vol. 24, pp. 565-567 Printed in the U.S.A All rights reserved.
* Oncology Intelligence GENE THERAPY TARGETED BY IONIZING RADIATION RALPH R. WEICHSELBAUM, M.D.,’ VIKAS P. SUKHATME, M.D.*
DENNIS E. HALLAHAN,
AND DONALD W. KUFE,
M.D.3
‘Department of RadiationandCellularOncology, University
of Chicago, Chicago,
M.D.,]
‘HowardHughes Medical Institute, Department IL; and ‘Dana Farber Cancer Center, Boston, MA
of Medicine,
Gene therapyused for the treatment of neoplastic diseases is not well localized. Ionizing radiation can be used to activate the transcription of exogenous genes that encode cytotoxic proteins. This may be accomplished through the use of radiation responsive elements distal to the transcription start site of such genes. We discuss this concept and describe a technique which can be used to amplify the signal initiated by localized radiation therapy. These techniques may be used to target gene therapy during the treatment of human neoplasms. Gene therapy, X-rays, Transcription.
INTRODUCTION
METHODS
AND
MATERIALS
The primary goal in radiation oncology has been the optimization of local and regional tumor cure rates. Therefore, most treatment strategies have been devised with local control as the primary objective. These strategies have included altered fractionation schemes, manipulating the tumor microenvironment, particle therapy, the addition of physical agents such as heat to radiation and combinations of radiotherapy and chemotherapy. In general, radiotherapy has not had a major impact on the treatment of systemic disease. Two examples of the use of ionizing radiation in the treatment of systemic disease, are total body irradiation as preparation for bone marrow transplantation and hemibody irradiation as primary treatment for multiple myeloma. It is likely that radioimmunotherapy will find a niche in the treatment of systemic disease due to more efficient tumor targeting and advances in radiochemistry and dosimetry. As an additional adjunct to the treatment of local and systemic disease, we propose the use of ionizing radiation to activate cytokine and/or toxin production. This can be accomplished by irradiating cells containing exogenous radiation-inducible DNA plasmids in which there are genes that encode cytotoxic agents or radioprotective factors. Gene therapy can thereby be targeted or localized by x-rays.
Ionizing radiation and gene regulation Studies in our laboratory revealed that radiation induces a cytotoxic factor in stationary phase cultures of some human soft tissue sarcoma cell lines. Biochemical analysis revealed that tumor necrosis factor a(TNF) was produced following irradiation (3). Tumor necrosis factor (Yis a cytokine with a wide range of actions including the lysis of transformed cells in vitro. Tumor necrosis factor cr killing is mediated through free radical formation and subsequent DNA and microtubule fragmentation (10, 11). Studies from our laboratory showed that TNF is a radiosensitizer of some human tumor cells (2, 3). Netta et al. demonstrated in vivo radioprotection of the hematopoietic compartment by TNF (6). Thus induction of TNF or other bioactive molecules by radiation may contribute to the biological effects of radiation. Radiation induced proteins may cause paracrine and endocrine effects on the host as well. Our findings have been reinforced by studies from the Memorial group which have shown that ionizing radiation induces the production of platelet derived growth factor (PDGF) (Yand basic fibroblast growth factor (bFGF) in vascular smooth muscle and endothelial cells. These investigators suggested that PDGF and FGF may contribute to the vascular component of the late effects of
Reprint request to: Ralph R. Weichselbaum, M.D., University of Chicago, Radiation and Cellular Oncology Department, 584 1 South Maryland, Box 442, Chicago, IL 60637. Acknowledgements-This work has been funded by: Center for Radiation Therapy, Chicago Tumor Institute, NIH Grants #CA-
41068 and #CA-42596, and the American Faculty Award (DEH). Accepted for publication 16 April 1992.
565
Cancer
Society Jr.
566
1. J. Radiation Oncology 0 Biology 0 Physics
radiotherapy on normal tissues by stimulating smooth muscle and endothelial cell proliferation (8). Because of the potential clinical usefulness of these cytokines and growth factors, we initiated studies to determine how radiation regulates gene expression. The activation of gene transcription by x-rays offers a potential means of manipulating genes that may increase the production of factors that cause enhancement of tumor cell killing or of other factors that may protect normal tissues against radiation injury. A detailed discussion of transcription factor activation is beyond the scope of this article. However, excellent reviews are available (5). In the context of this discussion, it is important to note that specific protein interactions occur between transcription factors bound to DNA elements and factors associated with the transcription initiation complex at RNA start sites. Proteins that bind to these specific DNA sequences may be activated in the absence of protein synthesis by a variety of mechanisms including protein phosphorylation, dephosphorylation and glycosylation. One potential mechanism of transcription factor activation following ionizing radiation exposure is through the increase in the phosphotransferase activity of protein kinase C which is observed immediately
Volume 24, Number 3. 1992
following irradiation (4). Protein kinase C also activates phosphatases which may enhance transcription factor binding to DNA. Radiation activated transcription factors directly initiate transcription of radiation inducible genes. In the case of TNF, radiation may activate Jun, NFKB, Egr-1 and possibly Fos (in myeloid cells) (4) and may cause combinations of these proteins to bind to specific DNA sequences in the promoter region of the TNF gene. We hypothesize for example, that a Jun-like DNA binding sequence S’TGAC/GTCA/3 or Egr-like binding sequence 5’-CGCCCCGC-3’ is involved in the binding of some radiation activated proteins. Investigation is underway to characterize the specific DNA sequences involved in transcription factor binding that initiate transcription following radiation exposure. Ionizing radiation and gene therapy
We propose that it may be possible to either regulate transcription of genes that encode cytotoxic proteins by placing radiation responsive Cis elements in front of genetic constructs which may activate or amplify radiation induced signals; or genes that encode proteins which increase radiation tolerance of dose limiting normal tissues. For example, a genetic construct with the VP-16 DNA
IONIZING RADIATION .
RADIATION
ENHANCEMENT
DNA BINDING
TARGET GENE
TRANSCRIPTION LAC REPRESSOR
Fig. 1. X-irradiation of Tumor Infiltrating Lymphocytes (TIL’s) that are transfected with a radiation inducible plasmid. A radiation responsive cis element (RRE) linked to a gene encoding a cytotoxic agent (e.g., tumor necrosis factor, TNF) will allow for the targeting of gene therapy to the irradiated volume.
Genetic radiotherapy 0
sequence that encodes a known powerful transactivating protein attached to the DNA coding sequence derived from the DNA binding domain for the Lac repressor might be inserted downstream of cis-acting elements which bind radiation inducible proteins (1). These constructs might be effective in amplifying radiation induced signals (Fig. 1). This construct could be co-transfected with the plasmid containing multiple DNA binding sites for the Lac repressor protein cloned upstream of genes ( 11) which when activated, alter the phenotypic response of tumors to radiation (e.g., tumor necrosis factor, growth factors, or toxins such as Ricin). A variety of technical obstacles are associated with this approach including the insertion of this genetic construct into appropriate cells and targeting these to tumors. However, Rosenberg’s group has placed the TNF gene in tumor infiltrating lymphocytes (TIL) and early success in gene therapy has been achieved in non-malignant disease (9). The pharmacological investigation of the distribution of toxins/cytokines as well as tumor infiltrating lymphocytes or other delivery vehicles will be necessary. The technology of gene manipulation applied to benign disease suggests a variety of approaches both for transfecting constructs into cells and targeting specific tissues in cancer patients (7). Targeting of gene therapy will be enhanced by the already well developed technology available in radiotherapy. For example, the patient may have lung metastases that could be treated with daily injections of TIL cells containing radiation inducible toxins. The cytotoxic agents
R.
R.
567
WEICHSELBAUM et ul.
will then be induced by daily doses of radiotherapy. Some cytotoxicity will occur from the radiation as well as the daily release of a radiosensitizing and/or tumor killing agent. It might even be possible to “cone down” to metastases in multiple organs depending upon the efficacy of the technology. Therefore, radiation will preferentially activate toxin production in the irradiated volume. This approach has advantages over LAK or TIL cells used alone because these activated lymphocytes may not provide highly specific targeting in spite of the best efforts of immunologic manipulation. This approach could also be applied to local disease where local intra-tumor radiation induction of a substance that sensitizes tumor cells to radiation and/or kills tumor cells might alter the therapeutic ratio favorably enough to increase local cure rates. It is particularly attractive to employ classes of cytokines such as TNF that give local tumor kill but protect the hematopoietic compartment from ionizing radiation. SUMMARY Genotoxic agents induce a variety of genes in bacteria and mammalian cells. Manipulation of the transcription factors that control these genes might allow regulation of therapeutically useful genes either in the context of radiosensitization or radioprotection of normal tissues. The advances in molecular biology might allow increased local control and place the radiotherapist at the center of the management of systemic disease.
REFERENCES 1. Bairn, S. B.; Labow, M. A.; Levine, A. J.; Shenk T. A chi-
2.
3.
4.
5.
6.
meric mammalian transactivator based on the lac repressor that is regulated by temperature and isopropyl beta-D-thiogalactopyranoside. Prcc. Natl. Acad. Sci. 88:5072-6076;199 1. Hallahan, D. E.; Beckett, M. A.; Kufe, D.; Weichselbaum, R. R. The interaction between recombinant human tumor necrosis factor and radiation in 13 human tumor cell lines. Int. J. Radiat. Oncol. Biol. Phys. 19:69-74;1990. Hallahan, D. E.; Spriggs, D. R.; Beckett, M. A.; Kufe, D. W.; Weichselbaum, R. R. Increased tumor necrosis factor alpha, mRNA after cellular exposure. Proc. Natl. Acad. Sci. 86:10104-10107;1989. Hallahan, D. E.; Sukhatme, V. P.; Sherman, M. L.; Virudachalam, S.; Kufe, D.; Weichselbaum, R. R. Protein kinaseC mediates inducibility of nuclear signal transducers EGR1 and c-JUN. Proc. Natl. Acad. Sci. 88:2 156-2 160; 199 1. Mitchel, P. J.; Tjian, R. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science 245:371-378;1989. Neta, R.; Oppenheim J. J.; Schreiber, R. D.; Chizzonite,
7. 8.
9.
10.
11.
R.; Ledney, G. D.; MacVittie, T. J. Role of cytokines (Interleukin 1,tumor necrosis factor, and transforming growth factor p) in natural and lipopolysaccharid-enhanced radioresistance. J. Exp. Med. 173: 1177- 1182;199 1. Verma, I. M. Gene therapy. Sci. Amer. 263:68-84;1990. Witte, L.; Fuks, Z.; Haimovitz-Friedman, A.; Vlodavsky, I.; Goodman, D. S.; Elder, A. Effects of radiation on the release of growth factors from cultured bovine, porcine and human endothelial cells. Cancer Res. 49:5066-5072;I 989. Wong, R. A.; Alexander, R. B.; Puri, R. K.; Rosenberg, S. A. In vivo proliferation of adoptively transferred tumorinfiltrating lymphocytes in mice. J. Immunother. 10: I20130;1991. Yamauchi, N.; Karizana, H.; Wantanabe, H.; Neda, H.; Maeda, M.; Nutus, Y. Intracellular hydroxyl radical production induced by recombinant human tumor necrosis factor. Cancer Res. 49:1671-1675;1989. Zimmerman, R. J.; Chart, A.; Leadon, S. A. Oxidative damage in murine tumor cells treated in vitro by recombinant TNF. Cancer Res. 49: 1644- 1648: 1989.
Inr. J Rodrarion Oncology Rio/. Phys, Vol. 24, pp. 565-567 Printed in the U.S.A All rights reserved.
* Oncology Intelligence GENE THERAPY TARGETED BY IONIZING RADIATION RALPH R. WEICHSELBAUM, M.D.,’ VIKAS P. SUKHATME, M.D.*
DENNIS E. HALLAHAN,
AND DONALD W. KUFE,
M.D.3
‘Department of RadiationandCellularOncology, University
of Chicago, Chicago,
M.D.,]
‘HowardHughes Medical Institute, Department IL; and ‘Dana Farber Cancer Center, Boston, MA
of Medicine,
Gene therapyused for the treatment of neoplastic diseases is not well localized. Ionizing radiation can be used to activate the transcription of exogenous genes that encode cytotoxic proteins. This may be accomplished through the use of radiation responsive elements distal to the transcription start site of such genes. We discuss this concept and describe a technique which can be used to amplify the signal initiated by localized radiation therapy. These techniques may be used to target gene therapy during the treatment of human neoplasms. Gene therapy, X-rays, Transcription.
INTRODUCTION
METHODS
AND
MATERIALS
The primary goal in radiation oncology has been the optimization of local and regional tumor cure rates. Therefore, most treatment strategies have been devised with local control as the primary objective. These strategies have included altered fractionation schemes, manipulating the tumor microenvironment, particle therapy, the addition of physical agents such as heat to radiation and combinations of radiotherapy and chemotherapy. In general, radiotherapy has not had a major impact on the treatment of systemic disease. Two examples of the use of ionizing radiation in the treatment of systemic disease, are total body irradiation as preparation for bone marrow transplantation and hemibody irradiation as primary treatment for multiple myeloma. It is likely that radioimmunotherapy will find a niche in the treatment of systemic disease due to more efficient tumor targeting and advances in radiochemistry and dosimetry. As an additional adjunct to the treatment of local and systemic disease, we propose the use of ionizing radiation to activate cytokine and/or toxin production. This can be accomplished by irradiating cells containing exogenous radiation-inducible DNA plasmids in which there are genes that encode cytotoxic agents or radioprotective factors. Gene therapy can thereby be targeted or localized by x-rays.
Ionizing radiation and gene regulation Studies in our laboratory revealed that radiation induces a cytotoxic factor in stationary phase cultures of some human soft tissue sarcoma cell lines. Biochemical analysis revealed that tumor necrosis factor a(TNF) was produced following irradiation (3). Tumor necrosis factor (Yis a cytokine with a wide range of actions including the lysis of transformed cells in vitro. Tumor necrosis factor cr killing is mediated through free radical formation and subsequent DNA and microtubule fragmentation (10, 11). Studies from our laboratory showed that TNF is a radiosensitizer of some human tumor cells (2, 3). Netta et al. demonstrated in vivo radioprotection of the hematopoietic compartment by TNF (6). Thus induction of TNF or other bioactive molecules by radiation may contribute to the biological effects of radiation. Radiation induced proteins may cause paracrine and endocrine effects on the host as well. Our findings have been reinforced by studies from the Memorial group which have shown that ionizing radiation induces the production of platelet derived growth factor (PDGF) (Yand basic fibroblast growth factor (bFGF) in vascular smooth muscle and endothelial cells. These investigators suggested that PDGF and FGF may contribute to the vascular component of the late effects of
Reprint request to: Ralph R. Weichselbaum, M.D., University of Chicago, Radiation and Cellular Oncology Department, 584 1 South Maryland, Box 442, Chicago, IL 60637. Acknowledgements-This work has been funded by: Center for Radiation Therapy, Chicago Tumor Institute, NIH Grants #CA-
41068 and #CA-42596, and the American Faculty Award (DEH). Accepted for publication 16 April 1992.
565
Cancer
Society Jr.
566
1. J. Radiation Oncology 0 Biology 0 Physics
radiotherapy on normal tissues by stimulating smooth muscle and endothelial cell proliferation (8). Because of the potential clinical usefulness of these cytokines and growth factors, we initiated studies to determine how radiation regulates gene expression. The activation of gene transcription by x-rays offers a potential means of manipulating genes that may increase the production of factors that cause enhancement of tumor cell killing or of other factors that may protect normal tissues against radiation injury. A detailed discussion of transcription factor activation is beyond the scope of this article. However, excellent reviews are available (5). In the context of this discussion, it is important to note that specific protein interactions occur between transcription factors bound to DNA elements and factors associated with the transcription initiation complex at RNA start sites. Proteins that bind to these specific DNA sequences may be activated in the absence of protein synthesis by a variety of mechanisms including protein phosphorylation, dephosphorylation and glycosylation. One potential mechanism of transcription factor activation following ionizing radiation exposure is through the increase in the phosphotransferase activity of protein kinase C which is observed immediately
Volume 24, Number 3. 1992
following irradiation (4). Protein kinase C also activates phosphatases which may enhance transcription factor binding to DNA. Radiation activated transcription factors directly initiate transcription of radiation inducible genes. In the case of TNF, radiation may activate Jun, NFKB, Egr-1 and possibly Fos (in myeloid cells) (4) and may cause combinations of these proteins to bind to specific DNA sequences in the promoter region of the TNF gene. We hypothesize for example, that a Jun-like DNA binding sequence S’TGAC/GTCA/3 or Egr-like binding sequence 5’-CGCCCCGC-3’ is involved in the binding of some radiation activated proteins. Investigation is underway to characterize the specific DNA sequences involved in transcription factor binding that initiate transcription following radiation exposure. Ionizing radiation and gene therapy
We propose that it may be possible to either regulate transcription of genes that encode cytotoxic proteins by placing radiation responsive Cis elements in front of genetic constructs which may activate or amplify radiation induced signals; or genes that encode proteins which increase radiation tolerance of dose limiting normal tissues. For example, a genetic construct with the VP-16 DNA
IONIZING RADIATION .
RADIATION
ENHANCEMENT
DNA BINDING
TARGET GENE
TRANSCRIPTION LAC REPRESSOR
Fig. 1. X-irradiation of Tumor Infiltrating Lymphocytes (TIL’s) that are transfected with a radiation inducible plasmid. A radiation responsive cis element (RRE) linked to a gene encoding a cytotoxic agent (e.g., tumor necrosis factor, TNF) will allow for the targeting of gene therapy to the irradiated volume.
Genetic radiotherapy 0
sequence that encodes a known powerful transactivating protein attached to the DNA coding sequence derived from the DNA binding domain for the Lac repressor might be inserted downstream of cis-acting elements which bind radiation inducible proteins (1). These constructs might be effective in amplifying radiation induced signals (Fig. 1). This construct could be co-transfected with the plasmid containing multiple DNA binding sites for the Lac repressor protein cloned upstream of genes ( 11) which when activated, alter the phenotypic response of tumors to radiation (e.g., tumor necrosis factor, growth factors, or toxins such as Ricin). A variety of technical obstacles are associated with this approach including the insertion of this genetic construct into appropriate cells and targeting these to tumors. However, Rosenberg’s group has placed the TNF gene in tumor infiltrating lymphocytes (TIL) and early success in gene therapy has been achieved in non-malignant disease (9). The pharmacological investigation of the distribution of toxins/cytokines as well as tumor infiltrating lymphocytes or other delivery vehicles will be necessary. The technology of gene manipulation applied to benign disease suggests a variety of approaches both for transfecting constructs into cells and targeting specific tissues in cancer patients (7). Targeting of gene therapy will be enhanced by the already well developed technology available in radiotherapy. For example, the patient may have lung metastases that could be treated with daily injections of TIL cells containing radiation inducible toxins. The cytotoxic agents
R.
R.
567
WEICHSELBAUM et ul.
will then be induced by daily doses of radiotherapy. Some cytotoxicity will occur from the radiation as well as the daily release of a radiosensitizing and/or tumor killing agent. It might even be possible to “cone down” to metastases in multiple organs depending upon the efficacy of the technology. Therefore, radiation will preferentially activate toxin production in the irradiated volume. This approach has advantages over LAK or TIL cells used alone because these activated lymphocytes may not provide highly specific targeting in spite of the best efforts of immunologic manipulation. This approach could also be applied to local disease where local intra-tumor radiation induction of a substance that sensitizes tumor cells to radiation and/or kills tumor cells might alter the therapeutic ratio favorably enough to increase local cure rates. It is particularly attractive to employ classes of cytokines such as TNF that give local tumor kill but protect the hematopoietic compartment from ionizing radiation. SUMMARY Genotoxic agents induce a variety of genes in bacteria and mammalian cells. Manipulation of the transcription factors that control these genes might allow regulation of therapeutically useful genes either in the context of radiosensitization or radioprotection of normal tissues. The advances in molecular biology might allow increased local control and place the radiotherapist at the center of the management of systemic disease.
REFERENCES 1. Bairn, S. B.; Labow, M. A.; Levine, A. J.; Shenk T. A chi-
2.
3.
4.
5.
6.
meric mammalian transactivator based on the lac repressor that is regulated by temperature and isopropyl beta-D-thiogalactopyranoside. Prcc. Natl. Acad. Sci. 88:5072-6076;199 1. Hallahan, D. E.; Beckett, M. A.; Kufe, D.; Weichselbaum, R. R. The interaction between recombinant human tumor necrosis factor and radiation in 13 human tumor cell lines. Int. J. Radiat. Oncol. Biol. Phys. 19:69-74;1990. Hallahan, D. E.; Spriggs, D. R.; Beckett, M. A.; Kufe, D. W.; Weichselbaum, R. R. Increased tumor necrosis factor alpha, mRNA after cellular exposure. Proc. Natl. Acad. Sci. 86:10104-10107;1989. Hallahan, D. E.; Sukhatme, V. P.; Sherman, M. L.; Virudachalam, S.; Kufe, D.; Weichselbaum, R. R. Protein kinaseC mediates inducibility of nuclear signal transducers EGR1 and c-JUN. Proc. Natl. Acad. Sci. 88:2 156-2 160; 199 1. Mitchel, P. J.; Tjian, R. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science 245:371-378;1989. Neta, R.; Oppenheim J. J.; Schreiber, R. D.; Chizzonite,
7. 8.
9.
10.
11.
R.; Ledney, G. D.; MacVittie, T. J. Role of cytokines (Interleukin 1,tumor necrosis factor, and transforming growth factor p) in natural and lipopolysaccharid-enhanced radioresistance. J. Exp. Med. 173: 1177- 1182;199 1. Verma, I. M. Gene therapy. Sci. Amer. 263:68-84;1990. Witte, L.; Fuks, Z.; Haimovitz-Friedman, A.; Vlodavsky, I.; Goodman, D. S.; Elder, A. Effects of radiation on the release of growth factors from cultured bovine, porcine and human endothelial cells. Cancer Res. 49:5066-5072;I 989. Wong, R. A.; Alexander, R. B.; Puri, R. K.; Rosenberg, S. A. In vivo proliferation of adoptively transferred tumorinfiltrating lymphocytes in mice. J. Immunother. 10: I20130;1991. Yamauchi, N.; Karizana, H.; Wantanabe, H.; Neda, H.; Maeda, M.; Nutus, Y. Intracellular hydroxyl radical production induced by recombinant human tumor necrosis factor. Cancer Res. 49:1671-1675;1989. Zimmerman, R. J.; Chart, A.; Leadon, S. A. Oxidative damage in murine tumor cells treated in vitro by recombinant TNF. Cancer Res. 49: 1644- 1648: 1989.